The transient receptor potential (TRP) cation channels are involved in thermosensation of mammals. These channels are located on sensory neurons of the dorsal root ganglion (DRG) or the trigeminal ganglion (TG). They detect and transduce thermal-stimuli into electrical signals, i.e., action potentials. The electrical signals are then propagated from peripheral tissues to the spinal cord and brain, where they are integrated and interpreted to trigger appropriate reflexive and cognitive responses (Jordt et al., Curr Opin Neurobiol. 2003, 13(4): 487-92). Distinct TRP channels that are activated by specific temperatures and expressed in specific patterns have been identified. The combinatorial effect of the TRP channels enables a mammal to sense a broad range of thermal stimuli, namely those that are uncomfortable, noxious, hot or cold. Accordingly, these thermal receptors represent highly promising targets for the treatment of various painful conditions or for uses in other conditions in which tissue cooling is desirable.
TRPM8, the transient receptor potential channel, melanostatin subfamily, type 8, also called CMR1, the cold-menthol receptor, is a nonselective cationic channel permeable to Ca2+. TRPM8 is expressed in a subpopulation of sensory neurons that is activated both by decreases in temperature and cooling compounds, such as menthol, eucalyptol, icilin (McKemy et al., Nature, 2002, 416:52-58). TRPM8 offers interesting insight into the fundamental biology of cold perception. Modulation of TRPM8 can be relevant for therapeutic applications. For example, cold treatment is often used as a method of pain relief. Since the TRPM8 receptor is responsive to cold and compounds, such as menthol and icilin, that mimic a cold-like sensation, it is anticipated that modulation of TRPM8 activity is relevant for therapeutic applications where cold or menthol treatment is used as a method of pain relief or other relief, such as congestive rhinitis, cough or asthmatic bronchitis. Modulation of function or expression of TRPM8 proteins can also be useful for patients having dermal or mucus membrane conditions, such as skin inflammation and dermal burns, including sunburn and razor burn, or sore throat. Modulation of TRPM8 activity can further be relevant in patients suffering from hypersensitivity to cold that causes cold allodynia. In addition, modulation of TRPM8 activity can also be relevant for treating acute pain, for example, toothache (odontalgia) and other trigeminally distributed pains, such as trigeminal neuralgia (tic douleureux) and temperomandibular joint pain. Since human TRPM8 has been identified as a marker that is associated with tumor growth (Tsavaler, L., et al. Cancer Res., 2002, 61:3760-3769), modulation of TRPM8 can also be useful for the diagnosis of various cellular proliferation disorders.
Like many ion channels, TRPM8 is regulated by calcium. For example, lowering extracellular Ca2+ concentrations in vivo (by infusion of EDTA) or immersing the channels in isolated perfused preparations strongly increases the activity of the receptor, while raising extracellular Ca2+ concentrations antagonizes the actions of menthol on spike frequency and burst firing pattern. In addition, a rise in intracellular Ca2+ concentrations, either from cooling or artificial induction, triggers cold adaptation, by shifting the temperature sensitivity of the cold- and menthol-activated current (Reid et al., J. Physiol., 2002, 545:595-614). Transient rises and falls in intracellular Ca2+ levels register as local or global Ca2+ sparks and control numerous physiological events. However, prolonged high Ca2+ presence in the cytoplasm kills the cells.
Calmodulin (CaM) is found to be involved in many signaling pathways to decode intracellular Ca2+ concentration levels. A variety of ion channels found in a wide range of species, from Homo to Paramecium, use calmodulin (CaM) as their constitutive or dissociable Ca2+-sensing subunits (Saimi et al., Annu Rev Physiol. 2002, 64:289-311). CaM is a small, acidic, and highly conserved soluble calcium binding protein that is ubiquitously expressed. CaM binds Ca2+ as a monomer with two pairs of EF hands, which are common calcium binding motifs. Upon binding Ca2+, CaM becomes more extended with each pair of its EF hands opening to reveal a hydrophobic patch that is available to bond with a target and “activate it.” (Saimi et al, supra). The activated targets include those involved in protein phosphorylation, cyclic nucleotide metabolism, calcium homeostasis, etc.
Some TRP proteins have been found to interact with calmodulin in a Ca2+-dependent manner (Tang et al., J. Biol. Chem. 2001, 276:21303-21310). For example, CaM-binding occurs at two domains within the cytoplasmic C-terminal region of the TRP4 protein (Trost et al., 2001, Biochem J. 355(Pt 3):663-70). The presence of a CaM-binding site in the C terminus of TRP3 has also been reported (Zhang et al., Proc Natl. Acad Sci USA. 2001, 98(6):3168-73).
There is a need for systems that can be used to identify and test compounds that potentially increase or decrease the activity of a TRPM8. Identification and testing of such compounds would enable the treatment of various disorders associated with chronic pain and for uses in other conditions in which tissue cooling is desirable.